Molecular and physiochemical evaluation of buck semen cryopreserved with antioxidants.

The current study evaluated the physiochemical quality and gene expression profile of post-thawed buck semen after supplementation with antioxidants [melatonin (M), L-carnitine (LC), cysteine (Cys), LC + M, M + Cys, LC + Cys, LC + Cys + M] in comparison with the non-treated control group. Physical and biochemical characteristics of semen were evaluated following freezing and thawing. Transcript abundance of six selected candidate genes was profile using quantitative real-time PCR. The data demonstrated significant enhancement of post-freezing total motility, progressive motility, percentage of live sperm, CASA parameters, plasma membrane and acrosome integrity in all groups supplemented with Cys, LC, M + Cys and LC + Cys compared with the control group. The biochemical analysis of semen indicated that semen groups supplemented with LC and LC + Cys recorded increased levels of GPX and SOD that were coupled with up-regulation of antioxidant genes (SOD1, GPX1 and NRF2) and mitochondrial transcripts (CPT2 and ATP5F1A). Moreover, H2O2 level and DNA fragmentation percentage were reduced compared with other groups. In conclusion, supplementation of Cys alone or in combination with LC positively improved the post-thaw physiochemical properties of rabbit semen through activation of bioenergetics-related mitochondrial genes and cellular antioxidant defence mechanism.

Co-expression of candidate genes regulating growth performance and carcass traits of Barki lambs in Egypt.

Sheep are considered one of the main sources of animal protein in Egypt and the producers of sheep mutton eagers to find biological criteria for selecting fast-growing lambs that reach market weight early. Therefore, the present study aimed to find a link between the expression profile of selected candidate genes with growth performance and carcass traits of Barki lambs. Thirty-eight Barki lambs were kept and fed individually after weaning till 12 months of age and were divided into 3 groups according to growth performance (fast, intermediate, and slow-growing). Three samples were taken from different body tissues (eye muscle, liver, and fat tail) of each group, directly during slaughtering and stored at - 80 °C until RNA isolation. Real-time PCR was used to profile selected candidate genes (RPL7, CTP1, FABP4, ADIPOQ, and CAPN3) and GAPDH was used as a housekeeping gene. The results indicated that the final body weight was significantly (P ≤ 0.05) greater in the fast (49.9 kg) and intermediate (40.7 kg) compared to slow-growing animals (30.8 kg). The hot carcass weight was heavier (P ≤ 0.05) in the fast and intermediate-growing (24.57 and 19.07 kg) than slow-growing lambs (15.10 kg). The blood profiles of T3 and T4 hormones in addition to other parameters such as total protein, total lipids, and calcium level showed no clear variations among different experimental groups. At the molecular level, our data demonstrated upregulation of genes involved in protein biosynthesis (RPL7), fatty acid oxidation (CPT1), and lipolysis (FABP4) in the fast and intermediate-growing lambs in all studied tissues which facilitate protein accretion, energy expenditure, and fatty acid partitioning required for muscle building up. Moreover, the expression profile of the gene involved in muscle development (CAPN3) was increased in fast and intermediate-growing compared to slow-growing lambs in order to support muscle proper development. On the other hand, a candidate gene involved in lipogenesis (ADIPOQ) was expressed similarly in fat and liver tissues; however, its expression was increased in muscles of fast and intermediate-growing lambs compared to slow-growing animals. In conclusion, the current study indicated that the expression profile of genes involved in metabolic activities of liver, muscle, and adipose tissue is linked with the growth performance of lambs although no variations were detected in blood parameters. This provides an evidence for the importance of co-expression of these genes in body tissues to determine the final body weight and carcass characteristics of Barki sheep.

Cellular and molecular alterations of buffalo oocytes cultured under two different levels of oxygen tension during in vitro maturation.

This study was conducted to monitor the cellular and molecular changes of buffalo cumulus-oocytes complexes (COCs) cultured under high or low oxygen levels. Morphologically good quality COCs (n = 1627) were screened using brilliant cresyl blue (BCB) staining and placed into three groups (BCB+, BCB- and control). All groups of COCs were cultured under low (5%) or high (20%) oxygen tensions. Intracellular and molecular changes including oocyte ultrastructure, lipid contents, mitochondrial activity and transcript abundance of genes regulating different pathways were analyzed in the matured oocyte groups. The results revealed that oxygen tension did not affect cumulus expansion rates, however the BCB+ group had a higher (P ≤ 0.05) expansion rate compared with the BCB- group. BCB- oocytes recorded the lowest meiotic progression rate (P ≤ 0.05) under high oxygen levels that was linked with an increased level of reactive oxygen species (ROS) compared with the BCB+ oocytes. Ultrastructure examination indicated that BCB+ oocytes had a higher rate of cortical granules migration compared with BCB- under low oxygen tension. In parallel, our results indicated the upregulation of NFE2L2 in groups of oocytes cultured under high oxygen tension that was coupled with reduced mitochondrial activity. In contrast, the expression levels of MAPK14 and CPT2 genes were increased (P ≤ 0.05) in groups of oocytes cultured under low compared with high oxygen tension that was subsequently associated with increased mitochondrial activity. In conclusion, data from the present investigation indicated that low oxygen tension is a favourable condition for maintaining the mitochondrial activity required for nuclear maturation of buffalo oocytes. However, low-quality oocytes (BCB-) responded negatively to high oxygen tension by reducing the expression of gene-regulating metabolic activity (CPT2). This action was an attempt by BCB- oocytes to reduce the increased levels of endogenously produced ROS that was coupled with decreased expression of the gene controlling meiotic progression (MAPK14) in addition to nuclear maturation rate.

Transcriptome profile and association study revealed STAT3 gene as a potential quality marker of bovine gametes.

The present study was aimed to investigate differences in molecular signatures in oocytes derived from Holstein-Friesian heifers with different genetic merit for fertility, euthanized during day 0 or day 12 of the estrous cycle. Moreover, association between single nucleotide polymorphisms (SNPs) of ODC1 and STAT3 genes and bull fertility traits was investigated. The gene expression patterns were analyzed using cDNA array and validated with quantitative real-time polymerase chain reaction (PCR). The result revealed that several genes have shown not only to be regulated by fertility merit but also by the day of oocyte recovery during the estrous cycle. The STAT3 gene was found to be upregulated in oocytes recovered from animals with high fertility merit at both day 0 and day 12. Some other genes like PTTG1, ODC1 and TUBA1C were downregulated at day 0 and upregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In contrast, the transcript abundance of TPM3 was upregulated at day 0 and downregulated at day 12 in high, compared with low, fertility merit recovered oocytes. In addition, ODC1 and STAT3 were found to be associated (P < 0.05) with sperm quality traits as well as flow cytometry parameters. Therefore, the expression of several candidate genes including ODC1 and STAT3 was related to the genetic merit of the cow. In addition polymorphisms in these two genes were found to be associated with bull semen quality.

Effect of flunixin meglumine and aspirin administration on conception rate and estrous cycle characteristics of Egyptian Baladi cows during hot season.

The current investigation aims to evaluate the effects of flunixin meglumine (FM) and aspirin as non-steroid anti-inflammatory drug (NSAID) administration on estrous cycles characteristics and conception rate of Egyptian Baladi cows during hot season. In the first phase, 30 cows were divided into 3 groups, 10 cows for each treatment. The first group was treated with FM at the rate of 1.1 mg/kg body weight (BW) intramuscular, while the second group was administrated aspirin solution orally at the rate of 50 mg/kg BW. The third group was assigned as control (CG) that has no treatment. The FM group was administrated on day 14 after mating, while aspirin was given on day 14 and day 15 post-mating. All cows were mated naturally after showing estrus signs. Pregnancy diagnosis was carried 60 days after mating by rectal palpation. In the second phase, cows were monitored for estrus behavior by visual observation twice a day. The length of normal estrous cycles was 20, 23, and 22 days in cows treated with FM, aspirin, and control cows, respectively. There was no significant effect of treatment on the length of normal estrous cycles in Egyptian cows (P < 0.05). Proportions of long cycles in Egyptian cows that treated with FM or aspirin and control were 75, 67.7, and 57.1%, respectively. Short cycles were completely absent in cows that treated with FM or aspirin, but it was 29% in CG. Mounting behavior and tail rising were not detected in CG compared to 0 and 33% in FM or 25 and 33% in aspirin treated cows, respectively. Conception or pregnancy rate were 60, 40, and 30%, respectively, in FM, aspirin treated, and CG. Treatment cows whether FM or aspirin group did not influence (P < 0.05) progesterone concentration during the 14 days and 21 days from estrous cycle in pregnant and non-pregnant Egyptian Baladi cows than CG. In conclusion, the results of this study clearly indicated beneficial effect of FM and aspirin administration on intense of displayed estrous behavior and conception rate of Egyptian Baladi cows during the hot season.

Integrated ovarian mRNA and miRNA transcriptome profiling characterizes the genetic basis of prolificacy traits in sheep (Ovis aries).

BACKGROUND: The highly prolific breeds of domestic sheep (Ovis aries) are globally valuable genetic resources for sheep industry. Genetic, nutritional and other environmental factors affect prolificacy traits in sheep. To improve our knowledge of the sheep prolificacy traits, we conducted mRNA-miRNA integrated profiling of ovarian tissues from two pure breeds with large (Finnsheep) vs. small (Texel) litter sizes and their F1 crosses, half of which were fed a flushing diet. RESULTS: Among the samples, 16,402 genes (60.6% known ovine genes) were expressed, 79 novel miRNAs were found, and a cluster of miRNAs on chromosome 18 was detected. The majority of the differentially expressed genes between breeds were upregulated in the Texel with low prolificacy, owing to the flushing diet effect, whereas a similar pattern was not detected in the Finnsheep. F1 ewes responded similarly to Finnsheep rather than displaying a performance intermediate between the two pure breeds. CONCLUSIONS: The identification and characterization of differentially expressed genes and miRNAs in the ovaries of sheep provided insights into genetic and environmental factors affecting prolificacy traits. The three genes (CST6, MEPE and HBB) that were differentially expressed between the group of Finnsheep and Texel ewes kept in normal diet appeared to be candidate genes of prolificacy traits and will require further validation.

Identification and characterization of miRNAs in the ovaries of a highly prolific sheep breed.

Until recently, there have been few studies concerning miRNAs or miRNA-mediated biological processes in sheep (Ovis aries). In the present study, we used a deep-sequencing approach to examine ovarian miRNAs and the mRNA transcriptomes in two ewes of a highly prolific breed, Finnsheep. We identified 113 known sheep miRNAs, 131 miRNAs conserved in other mammals and 60 novel miRNAs, the expression levels of which accounted for 78.22%, 21.73% and 0.05% of the total respectively. Furthermore, the 10 most abundantly expressed miRNAs in the two libraries were characterized in detail, and the putative target genes of these miRNAs were annotated using GO annotation and KEGG pathway enrichment analyses. Among the target genes, intracellular transducers (SMAD1, SMAD4, SMAD5 and SMAD9) and bone morphogenetic protein (BMP) receptors (BMPR1B and BMPR2) were involved in the transforming growth factor ß (TGFß) signaling pathway in the reproductive axis, and the most significant GO terms were intracellular part (GO:0044424), binding (GO:0005488) and biological_process (GO:0008150) for cellular component, molecular function and biological process respectively. Thus, these results expanded the sheep miRNA database and provided additional information on the prolificacy trait regulated through specific miRNAs in sheep and other mammals.

Developmental competence of equine oocytes: impacts of zona pellucida birefringence and maternally derived transcript expression.

In the present study, equine oocytes were classified into groups of presumably high and low developmental competence according to cumulus morphology, as well as oocyte ability to metabolise brilliant cresyl blue (BCB) stain. All oocytes were evaluated individually in terms of morphometry, zona pellucida birefringence (ZPB) and relative abundance of selected candidate genes. Oocytes with an expanded cumulus (Ex), representing those with presumably high developmental competence, had a significantly thicker zona (18.2 vs 17.3µm) and a significantly higher ZPB (64.6 vs 62.1) than oocytes with a compacted cumulus (Cp). Concurrently, oocytes classified as highly developmentally competent (BCB+) had a significantly thicker zona (18.8 vs 16.1µm) and significantly higher ZPB (63.1 vs 61.3) compared with oocytes classified as having low developmental competence. Expression of TFAM, STAT3 and CKS2 was significantly higher in Ex compared with Cp oocytes, whereas expression of COX1, ATPV6E and DNMT1 was lower. Together, the data reveal that developmentally competent equine oocytes are larger in size, have higher ZPB values and exhibit a typical genetic signature of maternally derived transcripts compared with oocytes with lower in vitro developmental competence.

Molecular, enzymatic responses and in vitro embryonic developmental competency of heat-shocked buffalo embryos co-cultured with granulosa cells monolayer.

The present study was designed to establish a suitable alternative approach to mitigate the adverse effect of high culture temperature on in vitro embryo development and the related molecular response in buffalo. Pre-cultured granulosa cells (GCs) were used as a monolayer during in vitro embryo culture until day 8 (day of fertilization = D0). Post fertilization, presumptive embryos were randomly assigned into two culture conditions: embryos cultured in the presence of GCs monolayer under normal culture temperature (N: 38.5 °C; GEN group) or heat shock (H: 40.5 °C; GEH group) and their counterpart groups of embryos cultured without GCs (EN and EH groups). Additionally, two groups of GCs monolayer were cultured without embryos up to day 8 under 38.5 °C (GN) or 40.5 °C (GH) for further spent culture media enzymatic analyses. Heat shock was administered for the first 2 h of culture then continued at 38.5 °C until day 8. The results indicated that under heat treatment, GCs enhanced (P ≤ 0.05) embryo cleavage and development (day 8) rates, which were comparable to the embryos cultured at 38.5 °C. On the molecular level, blastocysts of the GEH group showed similar expressions of metabolism-regulating genes (CPT2 and SlC2A1/GLUT1) and an antioxidant gene (SOD2) when compared to the blastocysts of the EN group. The relative expression of HSP90 was significantly up-regulated under heat shock and/or co-culture conditions. However, HSF1 expression was increased (P ≤ 0.05) in the GEH group. No statistical differences were observed among the study groups for the pluripotency gene NANOG, and stress resistance transcript NFE2L2. Regarding the enzymatic profile, the concentrations of SOD, total protein, and MDA were decreased (P ≤ 0.05) in the GEH group compared to the cultured GCs without embryos (GH group). In conclusion, GCs as a monolayer have a beneficial impact on alleviating heat stress at the zygote stage through the regulatory mechanisms of metabolic activity, defense system, and heat shock response genes.

Melatonin administration during the first half of pregnancy improves the reproductive performance of rabbits: Emphasis on ovarian and placental functions.

This study was designed to investigate the roles of melatonin administration during different sensitive windows of the first half of pregnancy in the function and gene expression of the ovary and placenta, hormone profile, and pregnancy outcomes in rabbits. Four equal experimental groups of 20 rabbits each were used. The first (FW), second (SW), and third (F + SW) groups comprised rabbits that orally received 0.7-mg melatonin/kg body weight during the first week, second weeks, and during both weeks of pregnancy; and the fourth group served as the control group (C). The total number of visible follicles significantly increased in all melatonin-treated groups compared with that in the C group. In all melatonin-treated groups, the number of absorbed fetuses was significantly reduced, whereas the weights of embryonic sacs and fetuses were higher than in the C group. The placenta efficiency was significantly increased in the F + SW group compared with that in the C group, followed by the SW group, whereas no significant difference in the placenta efficiency was found between the FW and C groups. Melatonin treatments significantly improved the expression of antioxidants, gonadotropin receptors, and cell cycle regulatory genes in the ovary, whereas only FW treatment upregulated steroidogenic acute regulatory gene. Compared with the C and FW groups, melatonin treatments during the SW and F + SW significantly upregulated the expression of most genes in the placenta. The concentrations of estradiol were significantly higher in the SW and F + SW groups than in the FW and C groups. The concentrations of progesterone were significantly increased in the FW group compared with those in the C and SW groups, whereas the F + SW group showed intermediate values. The litter size and weight at birth significantly increased in all melatonin-treated groups compared with those in the C group. The second week of pregnancy seems to be a sensitive window for melatonin actions during pregnancy. Thus, melatonin administration during the second week of pregnancy can be effective in improving pregnancy outcomes in rabbits.

Transcriptional response of the bovine endometrium and embryo to endometrial polymorphonuclear neutrophil infiltration as an indicator of subclinical inflammation of the uterine environment.

The aim of the present study was to analyse the effect of subclinical endometritis on endometrial and embryonic gene expression. A total of 49 cows at either Day 0 or Day 7 of the oestrous cycle (62-83 days post partum) following superovulation were classified as having subclinical endometritis (SE-0, SE-7) or a healthy endometrium (HE-0, HE-7) on the basis of endometrial cytological evaluation. Endometrial samples and associated embryos were subjected to global transcriptome analysis using the Bovine GeneChip (Affymetrix, Santa Clara, CA, USA) and aberrant transcript profiles were observed in SE-0 and SE-7 cows. At Day 0, 10 transcripts were found to be differentially expressed in endometrial samples. Specifically, the PDZK1, PXDN, DDHD2, GPLD1 and SULT1B1 genes were downregulated, whereas the PKIB, LOC534256, BT29392, LYZ and S100A14 genes were upregulated in SE-0 cows. Similarly, 11 transcripts were found to be differentially regulated on Day 7. Of these, GNPTG, BOLA-DQA5, CHD2, LOC541226, VCAM1 and ARHGEF2 were found to be downregulated, whereas PSTPIP2, BT236441 and MGC166084 were upregulated in SE-7 cows. Accordingly, endometrial health status affected the number of flushed, transferable embryos. In all, 20 genes were differentially regulated in blastocysts derived from HE-7 and SE-7 cows. Of these, GZMK, TCEAL4, MYL7, ADD3 and THEM50B were upregulated, whereas NUDCD2, MYO1E, BZW1, EHD4 and GZMB were downregulated. In conclusion, endometrial polymorphonuclear neutrophil infiltration as an indicator of subclinical endometritis is associated with changes in endometrial gene expression patterns, including genes involved in cell adhesion and immune modulation. Consequently, subclinical endometritis affects gene expression in embryos, including the expression of genes related to membrane stability, the cell cycle and apoptosis.

Post-thaw viability, developmental and molecular deviations in in vitro produced bovine embryos cultured with l-carnitine at different levels of fetal calf serum.

l-carnitine is a well-known an antioxidant that enhanced lipid metabolism. Therefore, this study investigated the influence of supplementing l-carnitine (LC) to in vitro culture medium on preimplantation development, quality, cryotolerance and transcription profile of candidate genes. Following in vitro fertilization, embryos at zygote stage were cultured with medium supplemented with LC at 1.5 mM and fetal calf serum (FCS) at 0, 2.5, 5, 7.5 and 10% of the CR1-aa culture media. Intracellular quality of produced embryos was measured using different fluorescent stains that measured reactive oxygen species (ROS), lipid and mitochondria intensities. In addition, total cell number and total apoptotic cells were counted per embryo. Quantitative expression of candidate genes was conducted to find out molecular response of embryos after treatment. Moreover, vitrification was done at day 8 of preimplantation development to evaluate post-thaw embryo viability. The results indicated improved blastocyst formation rate at day 8 of preimplantation development (day zero = day of IVF) when embryos cultured with LC supplementation at low FCS at levels of 2.5% (35.3%) and 5% (34.7%) compared to control (25.9%), LC + FCS 7.5% (26.5%) and LC + FCS 10% (28.1%) groups. The total number of blastocyst cells that were cultured with LC + FCS 2.5% and LC + FCS 5% was increased and the number of dead cells (apoptotic) was decreased compared to control counterparts. Intracellular mitochondria activity was enhanced and resulted in reduction of cytoplasmic lipid in embryos treated with LC + FCS 2.5% and LC + FCS 5% compared with other experimental embryo groups. In addition, intracellular reactive oxygen species level was reduced in LC + FCS 2.5%, LC + FCS 5% and LC + FCS 7.5% compared to control and LC + FCS 10% groups. The expression profile of genes regulating embryo quality (BCL2), metabolic activity (GLUT1, CPT2 and TFAM), lipolysis (LIPE, AMPKa1 and ACCα), resistance to stress (SOD2) and ability to induce pregnancy (IFNt) was up-regulated under low FCS (2.5% and 5%) combined with LC supplementation. On the other hand, genes regulating lipogenesis were down-regulated (ACSL3 and S1PR). It can be concluded that LC is an efficient culture media supplement when added with FCS at 2.5 and 5% which improved blastocyst development rate and quality. These improvements are due to enhanced utilization of intracellular embryo lipid that subsequently increased cryotolerance through orchestrating genes involved in various activities of bovine embryos.

Integrative QTL mapping and selection signatures in Groningen White Headed cattle inferred from whole-genome sequences.

Here, we aimed to identify and characterize genomic regions that differ between Groningen White Headed (GWH) breed and other cattle, and in particular to identify candidate genes associated with coat color and/or eye-protective phenotypes. Firstly, whole genome sequences of 170 animals from eight breeds were used to evaluate the genetic structure of the GWH in relation to other cattle breeds by carrying out principal components and model-based clustering analyses. Secondly, the candidate genomic regions were identified by integrating the findings from: a) a genome-wide association study using GWH, other white headed breeds (Hereford and Simmental), and breeds with a non-white headed phenotype (Dutch Friesian, Deep Red, Meuse-Rhine-Yssel, Dutch Belted, and Holstein Friesian); b) scans for specific signatures of selection in GWH cattle by comparison with four other Dutch traditional breeds (Dutch Friesian, Deep Red, Meuse-Rhine-Yssel and Dutch Belted) and the commercial Holstein Friesian; and c) detection of candidate genes identified via these approaches. The alignment of the filtered reads to the reference genome (ARS-UCD1.2) resulted in a mean depth of coverage of 8.7X. After variant calling, the lowest number of breed-specific variants was detected in Holstein Friesian (148,213), and the largest in Deep Red (558,909). By integrating the results, we identified five genomic regions under selection on BTA4 (70.2-71.3 Mb), BTA5 (10.0-19.7 Mb), BTA20 (10.0-19.9 and 20.0-22.7 Mb), and BTA25 (0.5-9.2 Mb). These regions contain positional and functional candidate genes associated with retinal degeneration (e.g., CWC27 and CLUAP1), ultraviolet protection (e.g., ERCC8), and pigmentation (e.g. PDE4D) which are probably associated with the GWH specific pigmentation and/or eye-protective phenotypes, e.g. Ambilateral Circumocular Pigmentation (ACOP). Our results will assist in characterizing the molecular basis of GWH phenotypes and the biological implications of its adaptation.

Adaptive and Biological Responses of Buffalo Granulosa Cells Exposed to Heat Stress under In Vitro Condition.

The steroidogenesis capacity and adaptive response of follicular granulosa cells (GCs) to heat stress were assessed together with the underlying regulating molecular mechanisms in Egyptian buffalo. In vitro cultured GCs were exposed to heat stress treatments at 39.5, 40.5, or 41.5 °C for the final 24 h of the culture period (7 days), while the control group was kept under normal conditions (37 °C). Comparable viability was observed between the control and heat-treated GCs at 39.5 and 40.5 °C. A higher release of E2, P4 and IGF-1 was observed in the 40.5 °C group compared with the 39.5 or 41.5 °C groups. The total antioxidant capacity was higher in response to heat stress at 39.5 °C. At 40.5 °C, a significant upregulation pattern was found in the expression of the stress resistance transcripts (SOD2 and NFE2L2) and of CPT2. The relative abundance of ATP5F1A was significantly downregulated for all heat-treated groups compared to the control, while TNFα was downregulated in GCs at 39.5 °C. Expression analyses of stress-related miRNAs (miR-1246, miR-181a and miR-27b) exhibited a significant downregulation in the 40.5 °C group compared to the control, whereas miR-708 was upregulated in the 39.5 and 40.5 °C groups. In conclusion, buffalo GCs exhibited different adaptive responses, to the different heat stress conditions. The integration mechanism between the molecular and secretory actions of the GCs cultured at 40.5 °C might provide possible insights into the biological mechanism through which buffalo GCs react to heat stress.

Physiological and molecular aspects of heat-treated cultured granulosa cells of Egyptian buffalo (Bubalus bubalis).

The physiological and molecular responses of granulosa cells (GCs) from buffalo follicles were investigated when there were in vitro heat stress conditions imposed. The cultured GCs were heat-treated at 40.5 °C for 24, 48 or 72 h while GCs of the control group were not heat-treated (37 °C). There were no differences in viability between control and heat-treated groups. There was an upward trend in increase in E2 secretion as the duration of heat stress advanced, being greater (P ≤ 0.05) for the GCs on which heat stress was imposed for 72 as compared with 24 h. In contrast, P4 release was less (P ≤ 0.05) from GCs heat-treated for 48 h than those cultured for 24 h and GCs of the control group. The relative abundance of ATP5F1A and SOD2 mRNA transcripts was consistent throughout the period when there was imposing of heat stress to sustain mitochondrial function. The relative abundance of CPT2 transcript was less in heat-treated GCs than in GCs of the control group. There was a greater relative abundance of SREBP1 and TNF-α mRNA transcripts after 48 h of heat-treatment of GCs than GCs of the control group. In conclusion, the results from the current study indicate buffalo GCs cultured when there was imposing of heat stress maintained normal viability, steroidogenesis and transcriptional profile. The stability of antioxidant status and increased transcription of genes regulating cholesterol biosynthesis and stress resistance may be defense mechanisms of buffalo GCs against heat stress.

In Vivo and In Vitro Evaluation of Bull Semen Processed with Zinc (Zn) Nanoparticles.

Defective sperms cause fertilization failure under both in vivo and in vitro conditions. Therefore, providing optimal conditions during semen storage is a prerequisite for maintaining viability. The current study investigated bull semen quality in vitro and in vivo when zinc (Zn) nanoparticles were used as antioxidant during semen processing and cryopreservation. In total, 32 ejaculates were collected from four Holstein bulls. All ejaculates were pooled and diluted with Bioxcell-extender containing 0 (control group), 10-6, 10-5, 10-4, 10-3, and 10-2 M of Zn nanoparticles. Several physical and biochemical sperm parameters were determined after freeze-thawing process. In vitro embryo development rate and pregnancy rate were monitored after in vitro fertilization or artificial insemination using semen treated with Zn nanoparticles. Plasma membrane integrity was improved (P < 0.05) in bull semen treated with 10-6 M (69.3%), and 10-2 (62.4%) of Zn nanoparticles compared to untreated group (51.3%). In addition, proportions of live spermatozoa with active mitochondria were increased (P < 0.05) in semen supplemented with Zn nanoparticles at concentration of 10-6 M (67.3%), and 10-2 (85.3%) compared to control group (49.8%). Moreover, the level of MDA was lower (P < 0.05) in semen with Zn nanoparticles at 10-6 M (2.97 mol/mL) and 10-2 (2.7 mol/mL) concentrations than control semen samples (3.77 mol/mL). However, sperm total and progressive motility, sperm viability, DNA fragmentation, and pregnancy rate were not affected by treatment of semen with Zn nanoparticles. On the other hand, supplementation of in vitro maturation media with 10-6 M Zn nanoparticles has increased blastocyst rate (P < 0.05) compared to other experimental groups, while addition of Zn nanoparticles-treated sperm during in vitro fertilization did not affect embryo development rate. In conclusion, supplementation of Zn nanoparticles to semen has improved its quality without affecting embryo development rate in vitro. However, in vitro embryo development rate was increased when Zn nanoparticles were supplemented to IVM media. This support the notion of Zn nanoparticles beneficial action on improving bovine gametes quality without affecting pregnancy rate.

Genetic Features of Reproductive Traits in Bovine and Buffalo: Lessons From Bovine to Buffalo.

Bovine and buffalo are important livestock species that have contributed to human lives for more than 1000 years. Improving fertility is very important to reduce the cost of production. In the current review, we classified reproductive traits into three categories: ovulation, breeding, and calving related traits. We systematically summarized the heritability estimates, molecular markers, and genomic selection (GS) for reproductive traits of bovine and buffalo. This review aimed to compile the heritability and genome-wide association studies (GWASs) related to reproductive traits in both bovine and buffalos and tried to highlight the possible disciplines which should benefit buffalo breeding. The estimates of heritability of reproductive traits ranged were from 0 to 0.57 and there were wide differences between the populations. For some specific traits, such as age of puberty (AOP) and calving difficulty (CD), the majority beef population presents relatively higher heritability than dairy cattle. Compared to bovine, genetic studies for buffalo reproductive traits are limited for age at first calving and calving interval traits. Several quantitative trait loci (QTLs), candidate genes, and SNPs associated with bovine reproductive traits were screened and identified by candidate gene methods and/or GWASs. The IGF1 and LEP pathways in addition to non-coding RNAs are highlighted due to their crucial relevance with reproductive traits. The distribution of QTLs related to various traits showed a great differences. Few GWAS have been performed so far on buffalo age at first calving, calving interval, and days open traits. In addition, we summarized the GS studies on bovine and buffalo reproductive traits and compared the accuracy between different reports. Taken together, GWAS and candidate gene approaches can help to understand the molecular genetic mechanisms of complex traits. Recently, GS has been used extensively and can be performed on multiple traits to improve the accuracy of prediction even for traits with low heritability, and can be combined with multi-omics for further analysis.

Bovine pretransfer endometrium and embryo transcriptome fingerprints as predictors of pregnancy success after embryo transfer.

Aberrant gene expression in the uterine endometrium and embryo has been the major causes of pregnancy failure in cattle. However, selecting cows having adequate endometrial receptivity and embryos of better developmental competence based on the gene expression pattern has been a greater challenge. To investigate whether pretransfer endometrial and embryo gene expression pattern has a direct relation with upcoming pregnancy success, we performed a global endometrial and embryo transcriptome analysis using endometrial and embryo biopsy technology and the pregnancy outcome information. For this, endometrial samples were collected from Simmental heifers at day 7 and 14 of the estrous cycle, one cycle prior to embryo transfer. In the next cycle, blastocyst stage embryos were transferred to recipients at day 7 of the estrous cycle after taking 30-40% of the blastocyst as a biopsy for transcriptome analysis. The results revealed that at day 7 of the estrous cycle, the endometrial gene expression pattern of heifers whose pregnancy resulting in calf delivery was significantly different compared with those resulting in no pregnancy. These differences were accompanied by qualitative and quantitative alteration of major biological process and molecular pathways. However, the transcriptome difference was minimal between the two groups of animals at day 14 of the estrous cycle. Similarly, the transcriptome analysis between embryos biopsies that resulted in calf delivery and those resulted in no pregnancy revealed a total of 70 differentially expressed genes. Among these, the transcript levels of 32 genes including SPAG17, PF6, UBE2D3P, DFNB31, AMD1, DTNBP1, and ARL8B were higher in embryo biopsies resulting in calf delivery. Therefore, the present study highlights the potential of pretransfer endometrial and embryo gene expression patterns as predictors of pregnancy success in cattle.

Identification and characterization of miRNAs expressed in the bovine ovary.

BACKGROUND: MicroRNAs are the major class of gene-regulating molecules playing diverse roles through sequence complementarity to target mRNAs at post-transcriptional level. Tightly regulated expression and interaction of a multitude of genes for ovarian folliculogenesis could be regulated by these miRNAs. Identification of them is the first step towards understanding miRNA-guided gene regulation in different biological functions. Despite increasing efforts in miRNAs identification across various species and diverse tissue types, little is known about bovine ovarian miRNAs. Here, we report the identification and characterization of miRNAs expressed in the bovine ovary through cloning, expression analysis and target prediction. RESULTS: The miRNA library (5'-independent ligation cloning method), which was constructed from bovine ovary in this study, revealed cloning of 50 known and 24 novel miRNAs. Among all identified miRNAs, 38 were found to be new for bovine and were derived from 43 distinct loci showing characteristic secondary structure. While 22 miRNAs precursor loci were found to be well conserved in more than one species, 16 were found to be bovine specific. Most of the miRNAs were cloned multiple times, in which let-7a, let-7b, let-7c, miR-21, miR-23b, miR-24, miR-27a, miR-126 and miR-143 were cloned 10, 28, 13, 4, 11, 7, 6, 4 and 11 times, respectively. Expression analysis of all new and some annotated miRNAs in different intra-ovarian structures and in other multiple tissues showed that some were present ubiquitously while others were differentially expressed among different tissue types. Bta-miR-29a was localized in the follicular cells at different developmental stages in the cyclic ovary. Bio-informatics prediction, screening and Gene Ontology analysis of miRNAs targets identified several biological processes and pathways underlying the ovarian function. CONCLUSION: Results of this study suggest the presence of miRNAs in the bovine ovary, thereby elucidate their potential role in regulating diverse molecular and physiological pathways underlying the ovarian functionality. This information will give insights into bovine ovarian miRNAs, which can be further characterized for their role in follicular development and female fertility as well.

Effect of the microenvironment and embryo density on developmental characteristics and gene expression profile of bovine preimplantative embryos cultured in vitro.

The Well of the Well (WOW) system has been developed to culture embryos in small groups or to track the development of single embryos. In the present study, we aimed to examine the effects of the microenvironment provided by the WOW system and embryo density on developmental rates, embryo quality and preimplantative gene expression profile of the resulting embryos. Embryos cultured in a group of 16 reached the blastocyst stage at a significantly lower level than zygotes cultured in a group of 50 (22.2 vs 30.3%), whereas zygotes cultured in WOW were able to compensate against low embryo densities, reaching a blastocyst rate as high as embryos cultured in a group of 50 (31.3 vs 30.3%). Moreover, embryos derived from WOW culture did not differ in terms of differential cell counts and apoptotic cell index compared with controls. The gene expression analysis revealed 62 transcripts to be upregulated and 33 transcripts to be downregulated by WOW culture. Comparing the in vivo derived blastocysts with the blastocysts derived from WOW culture, and group culture, expression of ATP5A1, PLAC8 and KRT8 was more similar to the embryos derived from WOW culture, whereas expression of S100A10 and ZP3 genes was more similar to blastocysts cultured in a group. In conclusion, microenvironment as well as embryo density significantly affected developmental rates. While subsequent blastocysts did not differ in terms of differential cell counts and apoptotic cell index, significant differences were observed in terms of the relative abundance of transcripts in the resulting embryos.